Loss attributable to crop diseases accounts for about 25-75% of the total production. For rice which is a major food crop in China, the annual plantation area is about 0.4 billion Mu. On the basis that the yield per Mu is 400 kg and the average reduction in output due to rice diseases is 10%, the annual economic loss will be as much as more than 30 billion RMB. As such, production of food crops is seriously threatened. So far, in addition to use of good seeds and modification of cultivation measures, plant diseases are mainly controlled by spraying chemical fungicides. Most of existing chemical fungicides are toxic and harmful to human body and animals to different degrees, and harmful components left over on the edible parts of plants result in potential threat to human health. This has received attention from the government and all levels of the society. Moreover, some chemical pesticides are hard to decompose, so that they are accumulated in the ecological system for a long period of time and pollute the environment, which is unfavorable to the sustainable developed of the social economy. Also, existing chemical pesticides are not very effective against some plant diseases. Therefore, while developing a new generation of chemical pesticides with endeavor, great efforts shall be put to study and develop high-performance biogenic pesticides that are safe, economic and highly compatible with the environment. At present, few types and amounts of biogenic pesticides are promoted and used in the production, and some types of biogenic pesticides result in resistance in plant pathogens due to long-time use, so that control efficiency is not ideal. In the case of rice sheath blight, pesticides for its control mainly depend on Jinggangmycin which is an old biogenic pesticide. However, after long-period use of nearly 40 years, some anastomosis groups of Rhizoctonia solani have become drug resistant. Moreover, Jinggangmycin is only effective against Rhizoctonia solani and has no obvious control effect against other pathogens, so it is greatly limited in respect of application scope.
A bio-pesticide growth-promoting antagonistic bacterium strain M18 has a high-efficiency, safe and broad-spectrum fungicidal effect on plant diseases. Also, it is well compatible with the environment and easily decomposed in the environment. The growth-promoting antagonistic bacterium strain M18 has been deposited at China General Microbiological Culture Collection Center, China Committee for Culture Collection of Microorganisms, a depository authority assigned by the State Intellectual Property Office of the PRC, the deposition No. being CGMCC NO.0462. Preparation and use of the bio-pesticide growth-promoting antagonistic bacterium M18 have been patented by the state with the patent no. ZL00119857.2. However, the bio-pesticide growth-promoting antagonistic bacterium strain M18 is a living bacterium agent whose mechanism mainly involves synthesis of an active component against plant diseases by strain M18 active bacteria to inhibit pathogens in crops, and the content of the synthesized active component is easily influenced by the metabolic regulation mechanism of the bacterium itself and environmental conditions. Therefore, M18 has a disadvantage of unstable control effect against plant diseases, so that it is hard for strain M18 to be applied in a large scale in agricultural production.
It has been proved that, the main active component of the growth-promoting antagonistic bacterium M18 for control of plant diseases is phenazine-1-carboxylic acid. Phenazine-1-carboxylic acid is extracted from the fermentation broth of growth-promoting antagonistic bacterium M18 to control crop diseases using the active component rather than the living bacterium, which also features high efficacy, safety, broad-spectrum and good compatibility with the environment. Also, this can overcome the deficiency that the growth-promoting antagonistic bacterium M18 is unstable in controlling diseases. However, synthesis of the active component phenazine-1-carboxylic acid through fermentation of the growth-promoting antagonistic bacterium M18 has a low titer which is only about 200 mg/L. How to increase fermentation titer and reduce cost becomes a bottleneck for development of this product. In recent years, we have made in-depth study on the regulation mechanism of synthesis of phenazine-1-carboxylic acid by the growth-promoting antagonistic bacterium M18 using molecular biological technologies. Based on this, through the means of genetic engineering, the two-component regulatory gene gacA in the genome of the growth-promoting antagonistic bacterium M18 is inactivated and mutated in a targeted manner, thereby obtaining M18G, a strain derived from M18. M18G greatly increases the yield of phenazine-1-carboxylic acid, so that the fermentation titer of phenazine-1-carboxylic acid reaches about 1500-1700 mg/L. The technical method of this study had been disclosed in Acta Microbiologica Sinica, Vol. 44, p 761-765 in 2004, and the title of the thesis is Differential Regulation of Phenazine-1-Carboxylic Acid and Pyoluteorin Production Mediated by Inactivated gacA in Pseudomonas sp. M18. In 2006, Chinese Patent (Patent No.: ZL200610023459.9) entitled Method for Preparation of Fungicides Using Growth-promoting Antagonistic Bacterium M18 provided a method for preparation of fungicides using growth-promoting antagonistic bacterium M18's derivative strains M18G and M18R, where the fungicide is prepared from the metabolites of microorganisms rather than living microorganisms, and the objective of increasing control effect is achieved by combination of the metabolites of the two derivative strains. In 2009, we further invented a method for production of phenazine-1-carboxylic acid by using bioengineering strain M18G carrying plasmid pME6032Phz, so that the fermentation titer of phenazine-1-carboxylic acid reaches 5700-6600 mg/L, which further reduces production cost and realizes large-scale application in agricultural production. This technology has been patented by the state with Patent No. ZL200910198664.2. Microorganism-originated fungicides with phenazine-1-carboxylic acid as a main component have been named Shenqinmycin by the Chinese pesticide denomination authority. Technical-grade Shenqinmycin and 1% Shenqinmycin suspension concentrate were granted with official registration nos. (Registration Nos.: PB20110314 and PB20110315) by the Ministry of Agriculture of the PRC in 2011. Shenqinmycin has been listed as one of the products promoted in the whole country during the 12th Five-Year Plan (Certificate No.: TG2011-002).
However, the control effect of phenazine-1-carboxylic acid against pathogens is closely related to the acidity (pH) under which it is used, and at pH 7.0, the antifungal activity of phenazine-1-carboxylic acid is only 20% of that at pH 5.0, so that the control effect of phenazine-1-carboxylic acid under alkaline conditions is greatly reduced. It is also found that, in the genomes of the growth-promoting antagonistic bacterium M18 and its derivative strain M18G, the phzH gene is a mutated and inactivated gene.